Generation of High-Yield, Functional Oligodendrocytes from a c-myc Immortalized Neural Cell Line, Endowed with Staminal Properties.

Mafalda Giovanna Reccia,Floriana Volpicelli,Luca Colucci-D'Amato,Eirkiur Benedikz,Åsa Fex Svenningsen

doi:10.3390/ijms22031124

Abstract

Neural stem cells represent a powerful tool to study molecules involved in pathophysiology of Nervous System and to discover new drugs. Although they can be cultured and expanded in vitro as a primary culture, their use is hampered by their heterogeneity and by the cost and time needed for their preparation. Here we report that mes-c-myc A1 cells (A1), a neural cell line, is endowed with staminal properties. Undifferentiated/proliferating and differentiated/non-proliferating A1 cells are able to generate neurospheres (Ns) in which gene expression parallels the original differentiation status. In fact, Ns derived from undifferentiated A1 cells express higher levels of Nestin, Kruppel-like factor 4 (Klf4) and glial fibrillary protein (GFAP), markers of stemness, while those obtained from differentiated A1 cells show higher levels of the neuronal marker beta III tubulin. Interestingly, Ns differentiation, by Epidermal Growth Factors (EGF) and Fibroblast Growth Factor 2 (bFGF) withdrawal, generates oligodendrocytes at high-yield as shown by the expression of markers, Galactosylceramidase (Gal-C) Neuron-Glial antigen 2 (NG2), Receptor-Interacting Protein (RIP) and Myelin Basic Protein (MBP). Finally, upon co-culture, Ns-A1-derived oligodendrocytes cause a redistribution of contactin-associated protein (Caspr/paranodin) protein on neuronal cells, as primary oligodendrocytes cultures, suggesting that they are able to form compact myelin. Thus, Ns-A1-derived oligodendrocytes may represent a time-saving and low-cost tool to study the pathophysiology of oligodendrocytes and to test new drugs.

Highlights

IntroductionNeural Stem Cells (NSCs) are undifferentiated cells endowed with self-renewal capacities and multipotent differentiation into the three major cell types of the Central Nervous
To investigate the effects of differentiation on gene expression, A1 cells were deprived of serum for 6 days and cAMP was added to the culture medium (Figure 1A, upper panel)
To assess whether long-term A1 neurosphere culture affected staminal and neural gene expression, we studied the mRNA expression of the staminal markers Nestin, Kruppel-like factor 4 (Klf4) and Sox2, the early neuronal marker β III tubulin, the astroglial marker glial fibrillary protein (GFAP) and the immature oligodendroglial marker GalC in primary, secondary and tertiary A1 neurosphere cultures

Summary

Introduction

Neural Stem Cells (NSCs) are undifferentiated cells endowed with self-renewal capacities and multipotent differentiation into the three major cell types of the Central Nervous. System (CNS), such as neurons, astrocytes and oligodendrocytes. NSCs play a critical role in the CNS development, homeostasis and repair and can be isolated and propagated from embryonic, neonatal and adult mammalian CNS. NSCs can be cultured and expanded as neurospheres, spherical cell clusters formed in a serum free medium supplemented with. EGF and/or FGF2 in a non-adhesive substrate. Upon mitogenic growth factors removal and adhesion to the substrate, NSCs undergo differentiation into neurons, astrocytes and oligodendrocytes [1,2,3]. NSC manipulation is a target of intense investigation either for therapeutic purposes or to better understand mechanisms underlying brain functions [4,5,6]

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